Impaired gephyrin G-domain trimerization and phase separation in a patient with developmental epileptic encephalopathy

Abstract

Epilepsy, a common neurological disorder is frequently linked to genetic variants in synaptic proteins. Here, we describe a de novo pathogenic missense variant in the gephyrin G-domain (G134R) identified in an epileptic patient with developmental delay and seizures. Functional analyses reveal that G134R disrupts higher-order oligomerization, leading to impaired liquid-liquid phase separation (LLPS) and synaptic clustering. Recombinant G134R-gephyrin variant forms lower oligomers with reduced molybdenum cofactor (Moco) synthesis. In non-neuronal cells, G134R fails to oligomerize beyond dimers and loses Moco synthesis function. In neurons, G134R is unable to form synaptic clusters and exerts a dominant-negative effect on WT-gephyrin, severely disrupting inhibitory synapse formation. Our findings highlight a critical role for the G-domain in gephyrin self-assembly and LLPS, shifting the focus from the E-domain-centric view of gephyrin function and providing a novel molecular mechanism for epilepsy linked to G-domain mutations.